Our long-term research goals are to understand the mechanisms that regulate stem cell fate decisions. This proposal focuses on epigenetic regulation of cell identity. Open chromatin has been proposed as one of the hallmarks of stem cells, explaining their multilineage potential. Using the hematopoietic stem cell (HSC) system as a model of adult stem cell differentiation, we propose to test whether HSC have a more open and dynamic chromatin structure than lineage committed progenitors and mature cells. In addition, we will test the functional consequences of chromatin condensation, and manipulate heterochromatin formation during HSC differentiation. Importantly, we have discovered that highly purified hematopoietic stem and progenitor cells have a more nuclease-sensitive chromatin structure compared to mature cells. This nuclease sensitivity correlates with linage potential and with differential three-dimensional chromatin organization. Here, we will investigate the mechanisms behind these differences. We will analyze the global molecular changes of DNA and histone proteins by sensitive assays capable of detecting differences in small numbers of cells. We will test how HSC differentiation and fate decisions change upon inhibition of chromatin modifying enzymes using a combination of complementary molecular, cellular, and functional assays in vitro and in vivo. Lastly, we will perform comparative chromatin analyses between HSC and leukemic stem cells and assess the contribution of chromatin modifications in leukemic transformation. We anticipate that these results will provide novel insights to the dynamics of chromatin structure and organization during stem cell differentiation and transformation, with important implications for both normal hematopoietic development and leukemogenesis.